Interferons (IFNs) have antiproliferative, antiviral and immunomodulatory activities. IFNs have been used to treat a number of tumors including renal cell carcinoma, chronic myelogenous leukemia, breast cancer. However, not all types of tumors are sensitive to the IFN-induced antiproliferative activity. The reason for the selective therapeutic effectiveness of IFNs on tumor cells is not known. IFN stimulation leads to the activation of JAK kinases, which then phosphorylates a family of latent cytoplasmic transcription factors named STATs (signal transducer and activator of transcription). Stat1 is essential for IFN-gamma-induced biological activity. We have recently shown that the constitutively active Stat1 can enhance IFN- gamma induced antiproliferative activity in NIH3T3 cells. In this proposal, I present the preliminary data which show that the IL-2 dependent human T cells are unresponsive to IFN-gamma due to the inhibition of Stat1 signaling pathway in these cells. Furthermore, overexpression of constitutively activated Stat1 protein in breast cancer cells failed to enhance the antiproliferative activity of IFN- gamma suggesting that other signaling pathways in addition to STAT may be involved in mediating the antiproliferative activity of IFN-gamma. The overall goal of this research proposal is to study the molecular mechanism of IFN-gamma responsiveness. First, I will study the molecular mechanism of IFN-gamma unresponsiveness in IL-2 dependent T cells. I will test if the inhibition of IFN-gamma responsiveness in these cells is due to the presence of constitutively expressed inhibitors of the JAK kinases: the SOCS (Suppresser of the Cytokine Signaling) proteins. The constitutively expressed, SOCS proteins in IL-2 dependent T cells will be cloned and tested for their ability to inhibit IFN-gamma response. I will also examine an alternative hypothesis that the inhibition of IFN-gamma response in the IL-2 dependent T-cells is a result of enhanced protein tyrosine phosphatase activity. Second, I will examine the effect of Stat1 signaling on IFN- gamma-mediated antiproliferative activity in a number of cell lines by overexpressing a constitutively activated mutant Stat1 protein. Other signaling pathways such as the Mitogen-Activated Protein Kinase (MAPK) and the Protein Kinase-C (PKC) pathways will be examined for their effects on IFN-gamma-induced antiproliferative activity. Results obtained from these experiments will improve our ability to design effective therapeutic strategies for tumors employing IFNs and expand our knowledge of the regulations of cell signaling in general.